Cyclic nucleotide-gated (CNG) ion channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. They are nonselective cation channels that are opened by the direct binding of cyclic nucleotides to the channel. The channels are highly specialized for their role in signal transduction. The long-term goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations. In the last several years, the allosteric activation and modulation of these channels has been shown to involve dynamic interactions between multiple domains of the channel: the cyclic nucleotide-binding domain (CNBD), the pore, the C-linker domain connecting the CNBD to the pore, and the amino-terminal region. However a number of fundamental questions still remain: What are the structural rearrangements in the CNBD and C-linker domains that are associated with channel opening? How do the subunit interactions change during gating? What are the structural rearrangements associated with Ca2+-calmodulin modulation of the channel? To address these and related questions, this grant will take particular advantage of two exciting new developments: 1) our solution of the x-ray crystal structure of the intracellular ligand binding and gating domains of the closely related HCN2 channel, and 2) our development of techniques for site-specific fluorescent labeling of the intracellular domain of CNG channels and recording fluorescence in cell-free membrane patches (termed patch-clamp fluorometry, PCF). The structure of the HCN2 channel reveals that the C-linker forms a novel tetramerization domain of the channel, situated between the CNBD and the pore. Furthermore, biochemical and electrophysiological experiments suggest that these subunit interactions are dynamic. These experiments will use mutational analysis and PCF to investigate the rearrangements in the CNG CNBD, C-linker, and amino-terminal region associated with channel activation and modulation. The results will provide insights into the mechanisms for the normal behavior of CNG channels and their malfunction in disease states.